Recombinant adenoviruses provide a versatile system for gene expression studies and therapeutic applications. We report herein a strategy that simplifies the generation and production of such viruses. A recombinant adenoviral plasmid is generated with a minimum of enzymatic manipulations, using homologous recombination in bacteria rather than in eukaryotic cells. After transfections of such plasmids into a mammalian packaging cell line, viral production is conveniently followed with the aid of green fluorescent protein, encoded by a gene incorporated into the viral backbone. Homogeneous viruses can be obtained from this procedure without plaque purification. This system should expedite the process of generating and testing recombinant adenoviruses for a variety of purposes.Recombinant adenoviruses currently are used for a variety of purposes, including gene transfer in vitro, vaccination in vivo, and gene therapy (1-4). Several features of adenovirus biology have made such viruses the vectors of choice for certain of these applications. For example, adenoviruses transfer genes to a broad spectrum of cell types, and gene transfer is not dependent on active cell division. Additionally, high titers of viruses and high levels of transgene expression generally can be obtained.Decades of study of adenovirus biology have resulted in a detailed picture of the viral life cycle and the functions of the majority of viral proteins (5, 6). The genome of the most commonly used human adenovirus (serotype 5) consists of a linear, 36-kb, double-stranded DNA molecule. Both strands are transcribed and nearly all transcripts are heavily spliced. Viral transcription units are conventionally referred to as early (E1, E2, E3, and E4) and late, depending on their temporal expression relative to the onset of viral DNA replication (6). The high density and complexity of the viral transcription units poses problems for recombinant manipulation, which therefore is usually restricted to specific regions, particularly E1, E2A, E3, and E4. In most recombinant vectors, transgenes are introduced in place of E1 or E3, the former supplied exogenously. The E1 deletion renders the viruses defective for replication and incapable of producing infectious viral particles in target cells; the E3 region encodes proteins involved in evading host immunity and is dispensable for viral production per se.Two approaches traditionally have been used to generate recombinant adenoviruses. The first involves direct ligation of DNA fragments of the adenoviral genome to restriction endonuclease fragments containing a transgene (7,8). The low efficiency of large fragment ligations and the scarcity of unique restriction sites have made this approach technically challenging. The second and more widely used method involves homologous recombination in mammalian cells capable of complementing defective adenoviruses (''packaging lines'') (9, 10). Homologous recombination results in a defective adenovirus that can replicate in the packaging line (e.g., 293 or 911 ce...

Through global profiling of genes that were expressed soon after p53 expression, we identified a novel gene termed PUMA (p53 upregulated modulator of apoptosis). The protein encoded by PUMA was found to be exclusively mitochondrial and to bind to Bcl-2 and Bcl-X(L) through a BH3 domain. Exogenous expression of PUMA resulted in an extremely rapid and profound apoptosis that occurred much earlier than that resulting from exogenous expression of p53. Based on its unique expression patterns, p53 dependence, and biochemical properties, PUMA may be a direct mediator of p53-associated apoptosis.

To assess the role of BAX in drug-induced apoptosis in human colorectal cancer cells, we generated cells that lack functional BAX genes. Such cells were partially resistant to the apoptotic effects of the chemotherapeutic agent 5-fluorouracil, but apoptosis was not abolished. In contrast, the absence of BAX completely abolished the apoptotic response to the chemopreventive agent sulindac and other nonsteroidal anti-inflammatory drugs (NSAIDs). NSAIDs inhibited the expression of the antiapoptotic protein Bcl-XL, resulting in an altered ratio of BAX to Bcl-XL and subsequent mitochondria-mediated cell death. These results establish an unambiguous role for BAX in apoptotic processes in human epithelial cancers and may have implications for cancer chemoprevention strategies.

Although several genes that might mediate p53-induced apoptosis have been proposed, none have previously been shown to play an essential role in this process through a rigorous gene disruption approach. We used a gene-targeting approach to evaluate p53-mediated death in human colorectal cancer cells. Expression of p53 in these cells induces growth arrest through transcriptional activation of the cyclin-dependent kinase inhibitor p21. If p21 is disrupted via gene targeting, the cells die through apoptosis. If the PUMA gene is also disrupted in such cells, apoptosis is prevented. The effects of PUMA on apoptosis were observed after exogenous overexpression of p53 as well as after exposure to hypoxia, a physiologic activator of p53, and DNA damage. The PUMA protein interacts with Bcl-X L and promotes mitochondrial translocation and multimerization of Bax. Accordingly, genetic disruption of BAX makes cells resistant to the apoptosis resulting from PUMA expression. These results suggest that the balance between PUMA and p21 is pivotal in determining the responses to p53 activation and provide a model for understanding the basis of p53 mutations in human cancer.